Filoviruses are enveloped, non-segmented, negative-strand RNA viruses of the virus family Filoviridae. Two members of this virus family have been identified to date: Marburg virus (MARV) and Ebola virus (EBOV). Filoviruses are extremely virulent, easily transmissible from person-to-person, and extraordinarily lethal, causing severe hemorrhagic fever in humans and non-human primates. Filovirus infections have a fatality rate in humans ranging from 23% to as high as 90%. Despite their transmissibility and lethality, however, no approved therapy or preventive vaccine is available.
During outbreaks, isolation of patients and use of protective clothing and disinfection procedures (together called viral hemorrhagic fever (VHF) isolation precautions or barrier nursing) has been sufficient to interrupt further transmission of Marburg or Ebola viruses, and thus to control and end the outbreak. Because there is no known effective treatment for the hemorrhagic fevers caused by filoviruses, transmission prevention through application of VHF isolation precautions is currently the only available means to control filovirus outbreaks.
The first filovirus was recognized in 1967 after a number of laboratory workers in Germany and Yugoslavia, who had been handling tissues from African green monkeys, developed severe hemorrhagic fever. A total of 31 cases and seven deaths were associated with these outbreaks. The virus was named Marburg virus (MARV) after Marburg, Germany, the site of one of the outbreaks. After the initial outbreaks the virus disappeared and did not reemerge until 1975, when a traveler, most likely exposed in Zimbabwe, became ill in Johannesburg, South Africa; the traveler's traveling companion and a nurse were also infected. A few sporadic cases of Marburg hemorrhagic fever (MHF) have been identified since that time, but the disease remains relatively rare.
The second filovirus, Ebola virus (EBOV), was first identified in 1976 when two outbreaks of Ebola hemorrhagic fever (EHF) occurred in northern Zaire (now the Democratic Republic of Congo) and southern Sudan. The outbreaks involved viruses which eventually proved to be two different species of Ebola virus, which were named after the nations in which they were discovered. Both viruses proved to be highly lethal, with 90% of the cases in Zaire and 50% of the cases in Sudan resulting in death. Since 1976, Ebola virus has appeared sporadically in Africa, with a few small- to medium-sized outbreaks confirmed between 1976 and 1979, and again in Gabon between 1994 and 1996. Larger epidemics of Ebola HF occurred in Kikwit, Zaire in 1995 and in Gulu, Uganda in 2000.
It appears that filoviruses are transmitted to humans from ongoing life cycles in one or more non-human animals. Despite numerous attempts to locate the natural reservoir or reservoirs of Ebola and Marburg viruses, however, their origins remain mysterious. Consequently, it also remains unclear just how the virus is transmitted from its natural reservoir(s) to humans. Once a human has been infected, however, further infections occur by person-to-person transmission. Specifically, transmission involves close personal contact between an infected individual or their body fluids and another person. During recorded outbreaks of hemorrhagic fever caused by filovirus infection, people who cared for (i.e., fed, washed, medicated) or worked very closely with infected individuals were especially at risk of becoming infected themselves. Nosocomial (hospital) transmission through contact with infected body fluids (i.e., via reuse of unsterilized syringes, needles, or other medical equipment contaminated with these fluids) has also been an important factor in the spread of disease. Minimizing close contact between uninfected and infected patients usually reduces the number of new filovirus infections in humans during an outbreak. Although filoviruses have displayed some capability of infection through small-particle aerosols in the laboratory, airborne spread among humans has not been clearly demonstrated.
Five strains of Ebola virus have been identified so far, and are named after their site of first appearance: Bundibugyo (BEBOV), Ivory Coast (EBOV-CdI, also called Tai Forest virus or TAFV), Reston (EBOV-Reston), Sudan (SEBOV), and Zaire (ZEBOV); the Zaire, Sudan, and Bundibugyo strains are commonly involved in morbidity and death in humans. Ebola-Reston is the only known filovirus that does not cause severe disease in humans, although it can be fatal in monkeys. Several strains of Marburg virus have been identified so far, with the Musoke strain having the highest lethality rate. See FIG. 1.
Structurally, filovirus virions may appear in several shapes, including long, sometimes branched filaments, as well as shorter filaments shaped like a “6”, the letter “U”, or a circle. Viral filaments can measure up to 14 micrometers (μm) in length, have a uniform diameter of 80 nanometers (nm), and are enveloped in a lipid membrane. Each virion contains one single-stranded, negative-sense RNA molecule approximately 19 kilobase pairs (kb) in length, which contains seven sequentially arranged genes in the order of nucleoprotein (NP), virion protein 35 (VP35), virion protein 40 (VP40), envelope glycoprotein (GP), virion protein 30 (VP30), virion protein 24 (VP24), and RNA-directed RNA polymerase protein (L). Upon entry into the host cell cytoplasm, the RNA is transcribed to generate polyadenylated, subgenomic mRNA species encoding the proteins. Transcription and translation lead to the synthesis of seven structural polypeptides, with presumed identical functions for each of the different filoviruses. Four proteins (NP, VP30, VP35 and L) are associated with the viral genomic RNA in the nucleocapsid complex. The three remaining structural proteins are membrane-associated; GP is a type I transmembrane protein, while VP24 and VP40 are probably located on the inner side of the membrane. The envelope glycoprotein (GP) appears in the viral envelope as a homotrimer (also referred to as a ‘peplomer’) comprising three copies of a heterodimer. The heterodimer contains two fragments of the full-length GP precursor (referred to as ‘GP0’) known as ‘GP1’ and ‘GP2’ produced by furin cleavage. GP1 and GP2 are linked by a disulfide bond. A non-structural, secreted glycoprotein (sGP) is expressed by EBOV, but not MARV (H. Feldmann & M. P. Kiley, Curr. Top. Microbiol. Immunol. 235:1-21 (1999)). New viral particles are created by budding from the surface of host cells (see below).
The filovirus life cycle begins with virion attachment to specific cell-surface receptors, followed by fusion of the virion envelope with cellular membranes and release of the virus nucleocapsid into the cytosol. The viral RNA-directed RNA polymerase (RNAP, also known as the ‘L’ protein) partially uncoats the nucleocapsid and transcribes the genes into positive-stranded mRNAs, which are then translated into structural and nonstructural proteins. See FIG. 2. The RNAP binds to a single promoter located at the 3′ end of the genome. Transcription either terminates after a gene or continues to the next gene downstream, meaning that genes close to the 3′ end of the genome are transcribed in the greatest abundance, while those towards the 5′ end of the genome are least likely to be transcribed. Gene order is therefore a simple but effective form of transcriptional regulation. The most abundant protein produced is the nucleoprotein (NP), cellular concentration of which determines when the RNAP switches from gene transcription to genome replication. Replication results in full-length, positive-stranded anti-genomes that are in turn transcribed into negative-stranded virus progeny genome copies. Newly synthesized structural proteins and genomes self-assemble and accumulate near the inside of the cell membrane. Virus particles are enveloped as they bud from the infected host cell, producing mature infectious virions.
Prior Vaccine Development
Many strategies have been evaluated during attempts to develop a safe, immunogenic vaccine capable of inducing protective immunity against infection by one or more filovirus species, with decidedly mixed results. An overview is summarized in Marzi and Feldmann (A. Marzi and H. Feldmann Expert Rev. Vaccines 13(4):521-531 (2014)). For instance, while a trivalent DNA vaccine comprising a mixture of three DNA plasmids, one expressing the envelope glycoprotein from ZEBOV, a second expressing the envelope glycoprotein from SEBOV, and a third expressing the nucleoprotein from ZEBOV was safe, immunogenic, and able to induce an antibody response against at least one of the three antigens in humans. CD8+ T-cell responses were detected in fewer than ⅓ of the vaccinated population (J. E. Martin et al., Clin. Vaccine Immunol. 13(11):1267-1277 (2006)). Similarly, a complex, pentavalent adenovirus-based ‘pan-filovirus’ vaccine comprising a mixture of four different recombinant adenoviruses expressing envelope glycoproteins from ZEBOV, SEBOV, Marburg-Ci67 (strain Ratayczak), Marburg-Musoke, and Marburg-Ravn, as well as nucleoproteins from ZEBOV and Marburg-Musoke, protected non-human primates from ZEBOV or MARV challenge and induced antibody responses to both types of virus, although it remains unclear whether the vaccine induced any CD8+ T-cell response (D. L. Swenson et al., Clin. Vaccine Immunol. 15(3):460-467 (2008)).
Intranasal administration of a recombinant paramyxovirus—human parainfluenza virus, serotype 3 (HPIV3)—expressing either the envelope glycoprotein or both the envelope glycoprotein and nucleoprotein from ZEBOV protected guinea pigs from subsequent challenge with EBOV. Rodent models are frequently poorly predictive of results in primates, with a number of previous EBOV vaccine candidates that were effective in rodents failing completely in non-human primates (A. Bukreyev et al., J. Virol. 80(5):2267-2279 (2006)). Intranasal administration of a recombinant HPIV3 expressing either the envelope glycoprotein or both the envelope glycoprotein and nucleoprotein from ZEBOV in rhesus monkeys showed that any construct expressing the envelope glycoprotein was moderately immunogenic and protected more than 80% of the animals against disease after post-vaccination challenge with ZEBOV (A. Bukreyev et al., J. Virol. 81(12):6379-6388 (2007)). Finally, a recombinant vesicular stomatitis virus (VSV) in which the VSV glycoprotein was replaced by the ZEBOV envelope glycoprotein protected 50% of guinea pigs, 100% of mice following treatment as late as 24 hours after an otherwise uniformly lethal infection. Four out of eight rhesus macaques (50%) were protected when treated 20 to 30 min after exposure providing a post-exposure treatment option for Ebola virus infection (H. Feldmann, PLoS Pathogens 3(1):54-61 (2007)).
Geisbert et al. evaluated the effects of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection in nonhuman primates. They used RNA replicon particles derived from an attenuated strain of Venezuelan equine virus (VEEV) expressing EBOV glycoprotein and nucleoprotein, recombinant Vaccinia virus (VACV) expressing EBOV glycoprotein, liposomes containing lipid A and inactivated EBOV, and a concentrated, inactivated whole-virion preparation. They found that none of these strategies successfully protected nonhuman primates from robust challenge with EBOV (T. H Geisbert et al., Emerging Infectious Diseases 8(3):503-507 (2002)).
Others have used Virus Like Particles (VLPs) expressed in mammalian, bacterial, plant or insect cells as non-replicating subunit vaccines (D. L. Swenson et al., Vaccine 23:3033-3042 (2005); K. L. Warfield et al., JID 196(2):430-437 (2007), N. Kushnir et al., Vaccine 31(1):58-83 (2012), K. L. Warfield ET AL., PLOS ONE 10(3):e0118881 (2015), K. L. Warfield and M. J. Aman JID 204:1053-1059 (2011), V. M. Wahl-Jensen et al., J Virol. 79(16):10442-10450 (2005), WO 2003/039477, WO 2006/046963, WO 2006/073422, WO 2004/042001, U.S. Pat. Nos. 8,900,595, 7,211,378) to induce antibody responses. However, filovirus VLPs require a cost-intensive and challenging production process and need to be stored at ambient temperature over time.
Thus, after expending considerable time and effort, a few promising vaccine candidates have emerged at preclinical stages, but at present no approved preventive vaccine is available. Given the transmissibility and lethality of filovirus infection, there is a pressing need for an effective vaccine.